CA2213266A1 - Method for isolation and purification of s-(1,2-dicarboxyethyl) glutathione - Google Patents
Method for isolation and purification of s-(1,2-dicarboxyethyl) glutathioneInfo
- Publication number
- CA2213266A1 CA2213266A1 CA002213266A CA2213266A CA2213266A1 CA 2213266 A1 CA2213266 A1 CA 2213266A1 CA 002213266 A CA002213266 A CA 002213266A CA 2213266 A CA2213266 A CA 2213266A CA 2213266 A1 CA2213266 A1 CA 2213266A1
- Authority
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- Canada
- Prior art keywords
- salt
- glutathione
- acid
- copper
- dicarboxyethyl
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
- C07K5/0215—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing natural amino acids, forming a peptide bond via their side chain functional group, e.g. epsilon-Lys, gamma-Glu
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Life Sciences & Earth Sciences (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
An improved method for isolating and purifying a glutathione derivative is disclosed. More particularly, the invention provides a method for isolating and purifying S-(1,2-dicarboxyethyl)glutathione or its pharmacologically acceptable salt from a reaction mixture available upon reacting glutathione or its salt with either fumaric acid or its salt or maleic acid or its salt, which comprises a first step of converting the S-(1,2-dicarboxyethyl)glutathione or salt thereof in the reaction mixture to thecorresponding copper salt, dissolving the copper salt in an aqueous solution of acetic acid, formic acid or propionic acid, and removing the contaminant glutathione, oxidized glutathione and fumaric acid copper salts with the aid of activated carbon and a second step of dissolving or suspending the isolated S-(1,2-dicarboxyethyl)glutathione copper salt in water and blowing hydrogen sulfide gas through the resulting aqueous solution or suspension to remove copper.
Description
METHOD FOR ISOLATION AND PURIFICATION
OF S-(l, 2-DICARBOXYETHYL)GLUTATHIONE
FIELD OF THE INVENTION
The present invention relates to a method for isolating and purifying a glutathione derivative. More particularly, the invention relates to a method for isolating and purifying S-(l, 2-dicarboxyethyl)glutathione or a pharmacologically acceptable salt thereof.
BACKGROUND OF THE INVENTION
PRIOR ART
S-(l, 2-dicarboxyethyl)glutathione (hereinafter referred to briefly as DCE-GS) is an acidic tripeptide, which D. H. Calam and S. G. Waley discovered in the bovine lens (Biochem. J., 86, 226, 1963). The subsequent research of Ohmori and his coworkers revealed that DCE-GS occurs in the liver and heart as well (Archives of Biochemistry and Biophysics Vol. 279, No. 1, May 15, pp. 146-150, 1990). With regard to its pharmacological profile, the inventors of the present invention have found, thus far, that this substance has anticoagulant activity (JP Kokai S63-8337), antithrombotic activity (JP Kokai Hl-79956), ~ntiinfl~mm~tory and/or antiallergic activity (JP Kokai H3-48626), and antihistaminic and hepatoprotectant activities (JP
Kokai H3-118334).
DCE-GS is readily available as glutathione and either fumaric acid or maleic acid are simply stirred together in water. However, it has been found difficult to recover DCE-GS in a high purified form from the reaction mixture on a high-production scale. The following three alternative methods have been proposed for isolating and purifying DCE-GS after completion of the reaction of glutathione with either fumaric acid or maleic acid.
One method for isolating and purifying DCE-GS comprises dissolving the reaction product free acid or salt in water, ~ing an organic solvent, such as an alcohol or acetone, to the solution to precipitate the product, and subjecting the precipitate to several cycles of recryst~lli7~tion (JP Kokai H3-118334). This method has the disadvantage that the residual starting materials, i.e. glutathione and fumaric acid, in the solution are also coprecipitated to cont~min~te DCE-GS, thus m~king it difficult to isolate DCE-GS in pure form.
Another method comprises converting the product DCE-GS to the corresponding copper salt and recryst~lli7.ing the salt from water-ethanol (JP
Kokai H3-118334). However, even by this method, it is difficult to thoroughly remove the cont~min~nt copper salts of glutathione, oxidized glutathione, and fumaric acid contained in small amounts.
In the method proposed by Waley et al., DCE-GS is first converted to the corresponding copper salt in the same manner as above and the copper is then elimin~ted with oxyquinoline. However, this method, too, is incapable of thorough purification of DCE-GS and, in addition, industrially disadvantageous in that it involves the use of the expensive reagent oxyquinoline and a complicated procedure (Biochem. J. 96, 226, 1963).
Thus, it is difficult, by any of the above known methods, to isolate the product DCE-GS in a highly purified form from the reaction mixture.
Therefore, the inventors of the present invention did much research to develop a commercially advantageous method for isolating and purifying DCE-GS. As a result, the inventors discovered that DCE-GS can be isolated in high purity by employing an aqueous solution of acetic acid, formic acid or propionic acid and activated carbon and have ultimately perfected the present invention.
OBJECT AND SUMMARY OF THE INVENTION
The present invention has for its object to provide an improved method for isolating and purifying DCE-GS, which is free of the above-mentioned disadvantages.
The present invention, therefore, is directed to a method for isolating and purifying S-(1, 2-dicarboxyethyl)glutathione or a ph~ cologically acceptable salt thereof from a reaction mixture available upon reacting glutathione or a salt thereof with either fumaric acid or a salt thereof or maleic acid or a salt thereof, which comprises a first step of converting the S-(l, 2-dicarboxyethyl)glutathione or salt thereof in said reaction rnixture to the corresponding copper salt, dissolving the copper salt in an aqueous solution of acetic acid, formic acid or propionic acid, and removing the cont~min~nt glutathione, oxidized glutathione and fumaric acid copper salts with the aid of activated carbon and a second step of dissolving or suspending the isolated S-(1, 2-dicarboxyethyl)glutathione copper salt in water and blowing hydrogen sulfide through the resulting aqueous solution or suspension to remove copper.
The method for isolating and purifying DCE-GS according to the present invention is now described in detail.
To start with, glutathione (or a salt thereof) and either fumaric acid (or a salt thereof) or maleic acid (or a salt thereof) are dissolved in water or an alcoholic aqueous medium and the solution is allowed to stand under warming or at room temperature for 1-2 days. By this procedure, the objective compound DCE-GS (or a salt thereof) is produced. The DCE-GS
(or a salt thereof) thus produced in the reaction mixture is converted to the corresponding copper salt using copper acetate (monohydrate) or the like.
After this copper salt is dissolved in an aqueous solution of acetic acid, formic acid or propionic acid, activated carbon is added to the solution to adsorb the copper salts of unreacted or excess glutathione, oxidized glutathione, and fumaric acid (in the reaction between maleic acid and glutathione, the unreacted maleic acid is transformed to fumaric acid) on the carbon and the carbon is then filtered off. Then, an alcohol or the like is added to the filtrate and the DCE-GS copper salt that separates out is recovered by filtration.
The DCE-GS copper salt thus obtained is dissolved or suspended in water and hydrogen sulfide gas is bubbled through the solution or suspension.
The copper, in the form of copper sulfide, is filtered off and the filtrate is concentrated under reduced pressure. To the oily residue is added an alcohol or the like and the resulting white crystals are collected by filtrationto provide DCE-GS in free acid form. When the oily residue is neutralized with an alkali metal or ~lk~line earth metal ion by adding the corresponding metal hydroxide, carbonate or hydrogen carbonate, the corresponding salt is obtained. This conversion to the salt may be performed after isolation of DCE-GS from the reaction mixture or in the reaction mixture. The objective compound DCE-GS or salt thereof is obtained in racemic form (since DCE-GS has asymmetric carbon within its molecule, it may occur as optical isomers). The purity of the product is not less than 98%.
The pharmacologically acceptable salt of DCE-GS includes but is not limited to salts with aLkali metals such as sodium, potassium, etc. and salts with ~lk~line earth metals such as calcium, magnesium, etc. The salt may be such that all of the carboxyl groups of DCE-GS have formed salts or only some of them have formed salts. DCE-GS in salt form shows a longer room-temperature shelf-life than DCE-GS in free acid form.
For recrystallizing the DCE-GS copper salt in the isolation and purification method of the present invention, the DCE-GS copper salt is first dissolved in an aqueous solution of acetic acid, formic acid or propionic acid before cryst~lli7.~tion from an alcohol. The presence of such an acid is advantageous in that the solubility of DCE-GS copper salt is increased and the cryst~lli7.~tion of DCE-GS from the alcohol is facilitated. In the absence of said acid, the DCE-GS copper salt on recryst~lli7~tion forms microfine crystals which cannot be easily collected by filtration.
In the above procedure for isolating and purifying DCE-GS, if hydrochloric acid or sulfuric acid, for instance, is used instead of acetic acid, formic acid or propionic acid for recryst~lli7~tion of DCE-GS copper salt, the copper chloride or copper sulfate salt will be formed in part, so that upon addition of an alcohol for precipitation of DCE-GS copper salt, the salt mentioned just above will also be coprecipitated. In the method for isolating and purifying DCE-GS according to the present invention, hydrogen sulfide gas is bubbled through the system to remove copper in the form of copper sulfide. However, it was found that some of the hydrochloric acid or sulfuric acid, it used, remains in the filtrate after removal of copper, withthe result that as the filtrate is concentrated, the concentration of the acid is increased to cause partial decomposition of the DCE-GS in the filtrate.
Furthermore, when the filtrate is neutralized, the hydrochloric acid or sulfuricacid forms an inorganic salt and when an alcohol is added, it is precipitated and cannot be easily removed. In contrast, when acetic acid, formic acid, or propionic acid is used as in the present invention, the above troubles are obviated so that DCE-GS can be isolated in a highly pure forrn.
The preferred concentration of the aqueous solution of acetic acid, formic acid or propionic acid for use in the isolation and purification method of the present invention for DCE-GS is invariably about 1-50 v/v %.
In the method for isolating and purifying DCE-GS according to the present invention, activated carbon is used for removing the copper salts of unreacted or excess glutathione or oxidized glutathione and furnaric acid. It has been found that without the aid of activated carbon, the starting glutathione and fumaric acid cannot be removed even by several cycles of recryst~lli7~tion. This is because the copper salts of glutathione and fumaric acid are less soluble than DCE-GS copper salt. The quantity of activated carbon for use in the method for isolating and purifying DCE-GS
according to the present invention with respect to S-(1, 2-dicarboxyethyl)glutathione copper salt is preferably about 5-20 w/w %.
EXAMPLES
The following examples are further illustrative of the present invention.
Example 1 Synthesis of S-(1, 2-dicarboxyethyl)glutathione trisodium salt In a 300 ml conical flask, 9.5 g of sodium hydroxide was dissolved in 200 ml of water, and 12.75 g (0.11 mol) offumaric acid and 30.7 g (0.1 mol) of glutathione were added. The flask was stoppered herrnetically and the contents were agitated at 50~C for 4 hours.
To this reaction mixture were added 50 ml of acetic acid and 22.0 g (0.11 mol) of copper acetate (monohydrate) with stirring. To the resulting solution was added about 3 g of activated carbon and the rnixture was stirred for 30 minutes and, then, ~ltered. The carbon was washed with a small quantity of water and the filtrate and washes were combined. After cooling, 1 L of methanol was added en bloc. The mixture was stirred until it became homogeneous and the precipitated light-blue copper salt was recovered by filtration, rinsed with methanol, and dried.
Then, for recryst~ ing the copper salt, it was dissolved in 400 ml of 10 v/v % aqueous solution of acetic acid and following addition of about 3 g of activated carbon, the mixture was stirred for 20-30 minutes. The carbon was then filtered off and lL of methanol was added en bloc to the cooled filtrate. The copper salt that separated out was recovered by filtration and rinsed with methanol. The copper salt was recrystallized again in the same manner as above to provide 40 g as copper salt.
This copper salt was dissolved (partially rem~ining undissolved) in 800 ml of water and hydrogen sulfide gas was bubbled through the solution with stirring. The resulting copper sulfide was filtered off and the filtrate was concentrated under reduced pressure at a temperature not exceeding 40~C. The residual syrup was dissolved in 200 ml of water, followed by dropwise addition of 10% sodium hydroxide to adjust the solution to pH 7.4.
The mixture was concentrated at 40~C and the residual syrup was crystallized from ethanol. After cooling, the crystal crop was recovered by filtration and recrystallized from water-ethanol. The crystals thus obtained were dried in vacuo at 40~C to provide 34 g (yield 65.8%) of the objective compound as white powdery crystals.
Elemental analysis for Cl4HI8OlON3SNa3 ~ 1.5H20 (516.366) Calcd.(%):C,32.56;H,4.10;N,8.14 Found (%): C, 32.67; H, 4.26; N, 8.13 By the isolation and purification technology of the present invention, DCE-GS or a salt thereof can be isolated in high purity from the reaction mixture available upon reacting glutathione or a salt thereof with fumaric acid (or a salt thereof) or maleic acid (or a salt thereof). Therefore, the technology of the invention is of great commercial value.
OF S-(l, 2-DICARBOXYETHYL)GLUTATHIONE
FIELD OF THE INVENTION
The present invention relates to a method for isolating and purifying a glutathione derivative. More particularly, the invention relates to a method for isolating and purifying S-(l, 2-dicarboxyethyl)glutathione or a pharmacologically acceptable salt thereof.
BACKGROUND OF THE INVENTION
PRIOR ART
S-(l, 2-dicarboxyethyl)glutathione (hereinafter referred to briefly as DCE-GS) is an acidic tripeptide, which D. H. Calam and S. G. Waley discovered in the bovine lens (Biochem. J., 86, 226, 1963). The subsequent research of Ohmori and his coworkers revealed that DCE-GS occurs in the liver and heart as well (Archives of Biochemistry and Biophysics Vol. 279, No. 1, May 15, pp. 146-150, 1990). With regard to its pharmacological profile, the inventors of the present invention have found, thus far, that this substance has anticoagulant activity (JP Kokai S63-8337), antithrombotic activity (JP Kokai Hl-79956), ~ntiinfl~mm~tory and/or antiallergic activity (JP Kokai H3-48626), and antihistaminic and hepatoprotectant activities (JP
Kokai H3-118334).
DCE-GS is readily available as glutathione and either fumaric acid or maleic acid are simply stirred together in water. However, it has been found difficult to recover DCE-GS in a high purified form from the reaction mixture on a high-production scale. The following three alternative methods have been proposed for isolating and purifying DCE-GS after completion of the reaction of glutathione with either fumaric acid or maleic acid.
One method for isolating and purifying DCE-GS comprises dissolving the reaction product free acid or salt in water, ~ing an organic solvent, such as an alcohol or acetone, to the solution to precipitate the product, and subjecting the precipitate to several cycles of recryst~lli7~tion (JP Kokai H3-118334). This method has the disadvantage that the residual starting materials, i.e. glutathione and fumaric acid, in the solution are also coprecipitated to cont~min~te DCE-GS, thus m~king it difficult to isolate DCE-GS in pure form.
Another method comprises converting the product DCE-GS to the corresponding copper salt and recryst~lli7.ing the salt from water-ethanol (JP
Kokai H3-118334). However, even by this method, it is difficult to thoroughly remove the cont~min~nt copper salts of glutathione, oxidized glutathione, and fumaric acid contained in small amounts.
In the method proposed by Waley et al., DCE-GS is first converted to the corresponding copper salt in the same manner as above and the copper is then elimin~ted with oxyquinoline. However, this method, too, is incapable of thorough purification of DCE-GS and, in addition, industrially disadvantageous in that it involves the use of the expensive reagent oxyquinoline and a complicated procedure (Biochem. J. 96, 226, 1963).
Thus, it is difficult, by any of the above known methods, to isolate the product DCE-GS in a highly purified form from the reaction mixture.
Therefore, the inventors of the present invention did much research to develop a commercially advantageous method for isolating and purifying DCE-GS. As a result, the inventors discovered that DCE-GS can be isolated in high purity by employing an aqueous solution of acetic acid, formic acid or propionic acid and activated carbon and have ultimately perfected the present invention.
OBJECT AND SUMMARY OF THE INVENTION
The present invention has for its object to provide an improved method for isolating and purifying DCE-GS, which is free of the above-mentioned disadvantages.
The present invention, therefore, is directed to a method for isolating and purifying S-(1, 2-dicarboxyethyl)glutathione or a ph~ cologically acceptable salt thereof from a reaction mixture available upon reacting glutathione or a salt thereof with either fumaric acid or a salt thereof or maleic acid or a salt thereof, which comprises a first step of converting the S-(l, 2-dicarboxyethyl)glutathione or salt thereof in said reaction rnixture to the corresponding copper salt, dissolving the copper salt in an aqueous solution of acetic acid, formic acid or propionic acid, and removing the cont~min~nt glutathione, oxidized glutathione and fumaric acid copper salts with the aid of activated carbon and a second step of dissolving or suspending the isolated S-(1, 2-dicarboxyethyl)glutathione copper salt in water and blowing hydrogen sulfide through the resulting aqueous solution or suspension to remove copper.
The method for isolating and purifying DCE-GS according to the present invention is now described in detail.
To start with, glutathione (or a salt thereof) and either fumaric acid (or a salt thereof) or maleic acid (or a salt thereof) are dissolved in water or an alcoholic aqueous medium and the solution is allowed to stand under warming or at room temperature for 1-2 days. By this procedure, the objective compound DCE-GS (or a salt thereof) is produced. The DCE-GS
(or a salt thereof) thus produced in the reaction mixture is converted to the corresponding copper salt using copper acetate (monohydrate) or the like.
After this copper salt is dissolved in an aqueous solution of acetic acid, formic acid or propionic acid, activated carbon is added to the solution to adsorb the copper salts of unreacted or excess glutathione, oxidized glutathione, and fumaric acid (in the reaction between maleic acid and glutathione, the unreacted maleic acid is transformed to fumaric acid) on the carbon and the carbon is then filtered off. Then, an alcohol or the like is added to the filtrate and the DCE-GS copper salt that separates out is recovered by filtration.
The DCE-GS copper salt thus obtained is dissolved or suspended in water and hydrogen sulfide gas is bubbled through the solution or suspension.
The copper, in the form of copper sulfide, is filtered off and the filtrate is concentrated under reduced pressure. To the oily residue is added an alcohol or the like and the resulting white crystals are collected by filtrationto provide DCE-GS in free acid form. When the oily residue is neutralized with an alkali metal or ~lk~line earth metal ion by adding the corresponding metal hydroxide, carbonate or hydrogen carbonate, the corresponding salt is obtained. This conversion to the salt may be performed after isolation of DCE-GS from the reaction mixture or in the reaction mixture. The objective compound DCE-GS or salt thereof is obtained in racemic form (since DCE-GS has asymmetric carbon within its molecule, it may occur as optical isomers). The purity of the product is not less than 98%.
The pharmacologically acceptable salt of DCE-GS includes but is not limited to salts with aLkali metals such as sodium, potassium, etc. and salts with ~lk~line earth metals such as calcium, magnesium, etc. The salt may be such that all of the carboxyl groups of DCE-GS have formed salts or only some of them have formed salts. DCE-GS in salt form shows a longer room-temperature shelf-life than DCE-GS in free acid form.
For recrystallizing the DCE-GS copper salt in the isolation and purification method of the present invention, the DCE-GS copper salt is first dissolved in an aqueous solution of acetic acid, formic acid or propionic acid before cryst~lli7.~tion from an alcohol. The presence of such an acid is advantageous in that the solubility of DCE-GS copper salt is increased and the cryst~lli7.~tion of DCE-GS from the alcohol is facilitated. In the absence of said acid, the DCE-GS copper salt on recryst~lli7~tion forms microfine crystals which cannot be easily collected by filtration.
In the above procedure for isolating and purifying DCE-GS, if hydrochloric acid or sulfuric acid, for instance, is used instead of acetic acid, formic acid or propionic acid for recryst~lli7~tion of DCE-GS copper salt, the copper chloride or copper sulfate salt will be formed in part, so that upon addition of an alcohol for precipitation of DCE-GS copper salt, the salt mentioned just above will also be coprecipitated. In the method for isolating and purifying DCE-GS according to the present invention, hydrogen sulfide gas is bubbled through the system to remove copper in the form of copper sulfide. However, it was found that some of the hydrochloric acid or sulfuric acid, it used, remains in the filtrate after removal of copper, withthe result that as the filtrate is concentrated, the concentration of the acid is increased to cause partial decomposition of the DCE-GS in the filtrate.
Furthermore, when the filtrate is neutralized, the hydrochloric acid or sulfuricacid forms an inorganic salt and when an alcohol is added, it is precipitated and cannot be easily removed. In contrast, when acetic acid, formic acid, or propionic acid is used as in the present invention, the above troubles are obviated so that DCE-GS can be isolated in a highly pure forrn.
The preferred concentration of the aqueous solution of acetic acid, formic acid or propionic acid for use in the isolation and purification method of the present invention for DCE-GS is invariably about 1-50 v/v %.
In the method for isolating and purifying DCE-GS according to the present invention, activated carbon is used for removing the copper salts of unreacted or excess glutathione or oxidized glutathione and furnaric acid. It has been found that without the aid of activated carbon, the starting glutathione and fumaric acid cannot be removed even by several cycles of recryst~lli7~tion. This is because the copper salts of glutathione and fumaric acid are less soluble than DCE-GS copper salt. The quantity of activated carbon for use in the method for isolating and purifying DCE-GS
according to the present invention with respect to S-(1, 2-dicarboxyethyl)glutathione copper salt is preferably about 5-20 w/w %.
EXAMPLES
The following examples are further illustrative of the present invention.
Example 1 Synthesis of S-(1, 2-dicarboxyethyl)glutathione trisodium salt In a 300 ml conical flask, 9.5 g of sodium hydroxide was dissolved in 200 ml of water, and 12.75 g (0.11 mol) offumaric acid and 30.7 g (0.1 mol) of glutathione were added. The flask was stoppered herrnetically and the contents were agitated at 50~C for 4 hours.
To this reaction mixture were added 50 ml of acetic acid and 22.0 g (0.11 mol) of copper acetate (monohydrate) with stirring. To the resulting solution was added about 3 g of activated carbon and the rnixture was stirred for 30 minutes and, then, ~ltered. The carbon was washed with a small quantity of water and the filtrate and washes were combined. After cooling, 1 L of methanol was added en bloc. The mixture was stirred until it became homogeneous and the precipitated light-blue copper salt was recovered by filtration, rinsed with methanol, and dried.
Then, for recryst~ ing the copper salt, it was dissolved in 400 ml of 10 v/v % aqueous solution of acetic acid and following addition of about 3 g of activated carbon, the mixture was stirred for 20-30 minutes. The carbon was then filtered off and lL of methanol was added en bloc to the cooled filtrate. The copper salt that separated out was recovered by filtration and rinsed with methanol. The copper salt was recrystallized again in the same manner as above to provide 40 g as copper salt.
This copper salt was dissolved (partially rem~ining undissolved) in 800 ml of water and hydrogen sulfide gas was bubbled through the solution with stirring. The resulting copper sulfide was filtered off and the filtrate was concentrated under reduced pressure at a temperature not exceeding 40~C. The residual syrup was dissolved in 200 ml of water, followed by dropwise addition of 10% sodium hydroxide to adjust the solution to pH 7.4.
The mixture was concentrated at 40~C and the residual syrup was crystallized from ethanol. After cooling, the crystal crop was recovered by filtration and recrystallized from water-ethanol. The crystals thus obtained were dried in vacuo at 40~C to provide 34 g (yield 65.8%) of the objective compound as white powdery crystals.
Elemental analysis for Cl4HI8OlON3SNa3 ~ 1.5H20 (516.366) Calcd.(%):C,32.56;H,4.10;N,8.14 Found (%): C, 32.67; H, 4.26; N, 8.13 By the isolation and purification technology of the present invention, DCE-GS or a salt thereof can be isolated in high purity from the reaction mixture available upon reacting glutathione or a salt thereof with fumaric acid (or a salt thereof) or maleic acid (or a salt thereof). Therefore, the technology of the invention is of great commercial value.
Claims (2)
1. A method for isolating and purifying S-(1,2-dicarboxyethyl)glutathione or a pharmacologically acceptable salt thereoffrom a reaction mixture available upon reacting glutathione or a salt thereof with either fumaric acid or a salt thereof or maleic acid or a salt thereof, which comprises a first step of converting the S-(1,2-dicarboxyethyl)glutathione or salt thereof in said reaction mixture to the corresponding copper salt, dissolving the copper salt in an aqueous solution of acetic acid, formic acid or propionic acid, and removing the contaminant glutathione, oxidized glutathione and fumaric acid copper salts with the aid of activated carbon and a second step of dissolving or suspending the isolated S-(1,2-dicarboxyethyl)glutathione copper salt in water and blowing hydrogen sulfide gas through the resulting aqueous solution or suspension to remove copper.
2. The method according to claim 1 wherein the activated carbon is used in a proportion of 5-20 w/w % with respect to said S-(1,2-dicarboxyethyl)glutathione copper salt and the concentration of said aqueous solution of acetic acid, formic acid, or propionic acid is 1-50 v/v %.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP233755/1996 | 1996-09-04 | ||
| JP23375596 | 1996-09-04 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2213266A1 true CA2213266A1 (en) | 1998-03-04 |
Family
ID=16960079
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002213266A Abandoned CA2213266A1 (en) | 1996-09-04 | 1997-08-18 | Method for isolation and purification of s-(1,2-dicarboxyethyl) glutathione |
Country Status (6)
| Country | Link |
|---|---|
| US (1) | US5773652A (en) |
| EP (1) | EP0827967B1 (en) |
| AT (1) | ATE176240T1 (en) |
| CA (1) | CA2213266A1 (en) |
| DE (1) | DE69700107T2 (en) |
| ES (1) | ES2127027T3 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB0520845D0 (en) * | 2005-10-14 | 2005-11-23 | Hofmann Thomas F | Salt enhancing compounds and use |
| CN113912671B (en) * | 2021-11-26 | 2024-04-19 | 江西诚志生物工程有限公司 | Glutathione separation and purification process |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5881797A (en) * | 1981-11-10 | 1983-05-17 | Kyowa Hakko Kogyo Co Ltd | Production of glutathione |
| JP2815179B2 (en) * | 1989-07-14 | 1998-10-27 | 千寿製薬株式会社 | Anti-inflammatory and / or anti-allergic agent |
| JP2919870B2 (en) * | 1989-09-29 | 1999-07-19 | 千寿製薬株式会社 | Liver damage inhibitor |
| EP0469156B1 (en) * | 1990-02-17 | 1996-10-23 | Senju Pharmaceutical Co., Ltd. | Glutathione derivative |
| TW207498B (en) * | 1991-02-27 | 1993-06-11 | Senju Pharma Co | |
| US5212159A (en) * | 1991-02-27 | 1993-05-18 | Senju Pharmaceutical Co., Ltd. | Anticataract composition |
-
1997
- 1997-08-18 CA CA002213266A patent/CA2213266A1/en not_active Abandoned
- 1997-08-30 DE DE69700107T patent/DE69700107T2/en not_active Expired - Fee Related
- 1997-08-30 ES ES97115073T patent/ES2127027T3/en not_active Expired - Lifetime
- 1997-08-30 EP EP97115073A patent/EP0827967B1/en not_active Expired - Lifetime
- 1997-08-30 AT AT97115073T patent/ATE176240T1/en not_active IP Right Cessation
- 1997-09-03 US US08/922,464 patent/US5773652A/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| ATE176240T1 (en) | 1999-02-15 |
| US5773652A (en) | 1998-06-30 |
| DE69700107D1 (en) | 1999-03-11 |
| ES2127027T3 (en) | 1999-04-01 |
| DE69700107T2 (en) | 1999-07-01 |
| EP0827967A1 (en) | 1998-03-11 |
| EP0827967B1 (en) | 1999-01-27 |
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